Mutations that confer genetic stability to genes in influenza viruses

Abstract

The disclosure provides for an isolated recombinant influenza virus having at least one of: a PB2 viral segment encoding PB2 with residue at position 540 that is not asparagine or a residue at position 712 that is not glutamic acid, a PA viral segment encoding PA with a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or a PB1 viral segment encoding PB1 with a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid or a residue at position 685 that is not aspartic acid, or any combination thereof, and methods of making and using the virus.

What is claimed is: 
     
       1. An isolated recombinant influenza virus having PA, PB1, PB2, NP, NS, M, NA, and HA viral segments, wherein i) at least one of the viral segments is a PB2 viral segment encoding PB2 with residue at position 540 that is not asparagine, a PA viral segment encoding PA with a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or a PB1 viral segment encoding PB1 with a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid or a residue at position 685 that is not aspartic acid, or any combination thereof, wherein the recombinant influenza virus has enhanced stability and/or enhanced replication relative to a corresponding recombinant influenza virus with a residue at position 540 in PB2 that is asparagine, a residue at position 180 in PA that is glutamine, a residue at position 200 in PA that is threonine, a residue at position 149 in PB1 that is valine, a residue at position 684 in PB1 that is glutamic acid or a residue at position 685 in PB1 that is aspartic acid; ii) at least one of the viral segments is a PB2 viral segment encoding PB2 with residue at position 540 that is not asparagine or a residue at position 712 that is not glutamic acid, and wherein at least one of the other viral segments is a PA viral segment encoding PA with a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or a PB1 viral segment encoding PB1 with a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid or a residue at position 685 that is not aspartic acid, or any combination thereof, wherein the recombinant influenza virus has enhanced stability and/or enhanced replication relative to a corresponding recombinant influenza virus with a residue at position 540 in PB2 that is asparagine, a residue in PB2 at position 712 that is glutamic acid, a residue at position 180 in PA that is glutamine, a residue at position 200 in PA that is threonine, a residue at position 149 in PB1 that is valine, a residue at position 684 in PB1 that is glutamic acid or a residue at position 685 in PB1 that is aspartic acid; or iii) the recombinant virus has two or more viral segments comprising a PB2 viral segment encoding PB2 with residue at position 540 that is not asparagine or a residue at position 712 that is not glutamic acid, a PA viral segment encoding PA with a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or a PB1 viral segment encoding PB1 with a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid or a residue at position 685 that is not aspartic acid, or any combination thereof, wherein the recombinant influenza virus has enhanced stability and/or enhanced replication relative to a corresponding recombinant influenza virus with a residue at position 540 in PB2 that is asparagine, a residue in PB2 at position 712 that is glutamic acid, a residue at position 180 in PA that is glutamine, a residue at position 200 in PA that is threonine, a residue at position 149 in PB1 that is valine, a residue at position 684 in PB1 that is glutamic acid or a residue at position 685 in PB1 that is aspartic acid. 
     
     
       2. The virus of  claim 1  wherein the residue at position 540 of PB2 is K, R, D, E, Q, or H, the residue at position 712 of PB2 is D, N, Q, S, H, T, Y, or C, the residue at position 180 in PA is R, K, D, E, N, or H, the residue at position 200 in PA is A, I, L, C, S, M, F, P, G, or V, the residue at position 149 in PB1 is A, T, I, L, C, S, M, F, P, or G, the residue at position 684 is D, Q, S, H, T, Y, C, K, R, or N, or the residue at position 685 in PB1 is E, N, R, H, K, S, T, Y, C, or Q; the residue at position 540 of PB2 is K, R, H, D, S, H, T, Y, or C, the residue at position 712 of PB2 is D, K, H, R, Q, or N, the residue at position 180 in PA is R, K, D, N, S, H, T, Y, or H, the residue at position 200 in PA is A, I, L, G, S, M, or V, the residue at position 149 in PB1 is A, T, I, L, S, M, or G, the residue at position 684 is D, Q, H, L, R or N, or the residue at position 685 in PB1 is E, N, R, H, K or Q; or the residue at position 540 of PB2 is K, R or H, the residue at position 712 of PB2 is D or N, the residue at position 180 in PA is R, K or H, the residue at position 200 in PA is A, I, L, G or V, the residue at position 149 in PB1 is A, T, I, L or G, the residue at position 684 is D or N, or the residue at position 685 in PB1 is E or Q. 
     
     
       3. The virus of  claim 1  wherein the PB2 has a residue at position 540 that is not asparagine, the PA has a residue at position 180 that is not glutamine and a residue at position 200 that is not threonine, and the PB1 has a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid or a residue at position 685 that is not aspartic acid; the PB2 has a residue at position 540 that is not asparagine, the PA has a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, and the PB1 has a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid or a residue at position 685 that is not aspartic acid; the PB2 has a residue at position 540 that is not asparagine or a residue at 712 that is not aspartic acid, the PA has a residue at position 180 that is not glutamine and a residue at position 200 that is not threonine, and the PB1 has a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid or a residue at position 685 that is not aspartic acid; or the PB2 has a residue at position 540 that is not asparagine and a residue at 712 that is not aspartic acid, the PA has a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, and the PB1 has a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid or a residue at position 685 that is not aspartic acid. 
     
     
       4. The virus of  claim 1  wherein the PA further comprises a residue at position 443 that is not arginine, the PB1 further comprises a residue at position 737 that is not lysine, the PB2 further comprises a residue at position 25 that is not valine or a residue at position 712 that is not glutamic acid, the NS viral segment encodes a NS1 with a residue at position 167 that is not proline, the HA viral segment encodes a HA with a residue at position 380 that is not threonine, or any combination thereof. 
     
     
       5. The virus of  claim 4  wherein the residue at position 443 of PA is K or H, the residue at position 737 of PB1 is H or R, the residue at position 25 of PB2 is A, L, T, I, or G, the residue at position 712 of PB2 is D, the residue at position 167 of NS1 is S, C, M, A, L, I, G or T, or any combination thereof. 
     
     
       6. The virus of  claim 1  wherein at least one of the viral segments includes a heterologous gene sequence encoding a gene product. 
     
     
       7. The recombinant virus of  claim 6  wherein the heterologous sequence is in the NS viral segment, M viral segment, NP viral segment, PA viral segment, PB1 viral segment, or the PB2 viral segment. 
     
     
       8. A vaccine having the isolated recombinant virus of  claim 1 . 
     
     
       9. The vaccine of  claim 8  wherein the virus encodes a non-influenza microbial protein, a heterologous influenza protein or a cancer associated antigen. 
     
     
       10. A plurality of influenza virus vectors for preparing a reassortant, comprising
 a) a vector for vRNA production comprising a promoter operably linked to an influenza virus PA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus PB1 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus PB2 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus HA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus NP DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus M DNA linked to a transcription termination sequence, and a vector for vRNA production comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, wherein the PB1, PB2, or PA DNAs in the vectors for vRNA production encode at least one of: a PB2 viral segment encoding PB2 with residue at position 540 that is not asparagine, a PA viral segment encoding PA with a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or a PB1 viral segment encoding PB1 with a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid or a residue at position 685 that is not aspartic acid, or a combination thereof; and optionally 
 b) a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M2, or a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS2. 
 
     
     
       11. The vectors of  claim 10  wherein the PB1, PB2, PA, NP, NS, and M DNAs in the vectors for vRNA production have a sequence corresponding to one that encodes a polypeptide having at least 95% amino acid sequence identity to a corresponding polypeptide encoded by SEQ ID NOs:1-6 or 10-15. 
     
     
       12. The vectors of  claim 10  wherein the residue at position 540 of PB2 is K, R or H, the residue at position 180 in PA is R, K or H, the residue at position 200 in PA is A, I, L, G or V, the residue at position 149 in PB1 is A, T, I, L or G, the residue at position 684 is D or N, or the residue at position 685 in PB1 is E or Q. 
     
     
       13. The vectors of  claim 10  wherein at least one of the viral segments includes a heterologous gene sequence encoding a gene product. 
     
     
       14. A method to prepare influenza virus, comprising: contacting a cell with:
 a vector for vRNA production comprising a promoter operably linked to an influenza virus PA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus PB1 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus PB2 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus HA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus NP DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus M DNA linked to a transcription termination sequence, and a vector for vRNA production comprising a promoter operably linked to an influenza virus NS DNA linked to a transcription termination sequence, wherein the PB1, PB2, or PA DNAs in the vectors for vRNA production encode 
 i) a PB2 with residue at position 540 that is not asparagine or a residue at position 712 that is not glutamic acid, and at least one: a PA with a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or a PB1 with a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid or a residue at position 685 that is not aspartic acid, or any combination thereof, ii) a PB2 with residue at position 540 that is not asparagine, a PA with a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or a PB1 with a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid or a residue at position 685 that is not aspartic acid, or any combination thereof, or iii) two or more of: a PB2 with residue at position 540 that is not asparagine or a residue at position 712 that is not glutamic acid, a PA with a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or a PB1 with a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid or a residue at position 685 that is not aspartic acid, or any combination thereof; and optionally 
 a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M2, or a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS2; 
 in an amount effective to yield infectious influenza virus. 
 
     
     
       15. The method of  claim 14  wherein the cell is an avian cell or a mammalian cell. 
     
     
       16. The method of  claim 15  wherein the cell is a Vero cell, a human cell or a MDCK cell. 
     
     
       17. The method of  claim 14  wherein the wherein the PB1, PB2, PA, NP, NS, and M DNAs in the vectors for vRNA productions have a sequence that corresponds to one that encodes a polypeptide having at least 95% amino acid sequence identity to a corresponding polypeptide encoded by SEQ ID NOs:1-6 or 10-15. 
     
     
       18. The method of  claim 14  wherein the residue at position 540 of PB2 is K, R or H, the residue at position 712 of PB2 is D or N, the residue at position 180 in PA is R, K or H, the residue at position 200 in PA is A, I, L, G or V, the residue at position 149 in PB1 is A, T, I, L or G, the residue at position 684 is D or N, or the residue at position 685 in PB1 is E or Q. 
     
     
       19. The method of  claim 14  wherein the influenza virus includes a heterologous gene sequence encoding a gene product. 
     
     
       20. The method of  claim 19  wherein the heterologous sequence is 5′ or 3′ to the PA coding sequence in the PA viral segment, 5′ or 3′ to the PB1 coding sequence in the PB1 viral segment, 5′ or 3′ to the PB2 coding sequence in the PB2 viral segment or 5′ or 3′ to the NS1 coding sequence in the NS viral segment.